The complete description of protein dynamics requires three variables: times scales, amplitudes, and spatial extent (McCammon & Harvey, 1987). Stopped-flow instruments with fluorescence and CD capabilities have been widely used to determine the rate and number of steps involved in protein folding reactions with considerable success. However, these experiments failed to give the spatial information of the protein during the folding/unfolding reactions. In this study we apply the method of fluorescence energy transfer combined with stopped-flow technique to follow global structural changes during folding of a staphylococcal nuclease mutant K45C, where lysine 45 was replaced by a cysteine. We labeled the thiol group with DTNB (5,5'-dithiobis(2-nitrobenzoic acid)) and used the TNB group cov...[ Read more ]

The complete description of protein dynamics requires three variables: times scales, amplitudes, and spatial extent (McCammon & Harvey, 1987). Stopped-flow instruments with fluorescence and CD capabilities have been widely used to determine the rate and number of steps involved in protein folding reactions with considerable success. However, these experiments failed to give the spatial information of the protein during the folding/unfolding reactions. In this study we apply the method of fluorescence energy transfer combined with stopped-flow technique to follow global structural changes during folding of a staphylococcal nuclease mutant K45C, where lysine 45 was replaced by a cysteine. We labeled the thiol group with DTNB (5,5'-dithiobis(2-nitrobenzoic acid)) and used the TNB group covalently attached to thiol group as an energy acceptor from a single tryptophan at residues 140 as the donor. Distance changes between the donor-acceptor pair during the folding reaction was estimated from the efficiency of energy transfer between the pair. The results support the idea that the denatured state of SNase is highly compact. The distance between the two probes increased from around 20Å to 25Å when the protein was unfolded, and the dimensions of acid-induced and GdmCl-induced denatured states have no observable differences. The fitting of the changes of distance shows that the folding reaction consists of three kinetic phases, and the unfolding reaction is a single phase. These results agree with the sequential model of Su et al. (1996) D₃↔D₂↔D₁↔ N₀, and provides information on the global conformations these denatured states.